This invention relates to an improved affinity gel-adsorbent for use in affinity chromatography. More particularly, this invention relates to an improved affinity gel-adsorbent for use in an improved method of purifying glucocorticoid receptor by affinity chromatography, said affinity gel-adsorbent having the above formula.
Cytosol is an extract of a cell. In the present case, it is a glucocorticoid receptor extract of a rat liver in the unactivated form or state which contains a plurality of proteins.
The involvement of proteins for glucocorticoids in intracellular action has been intensively studied during the past decade. However, basic to these studies is a knowledge of the molecular activities of these proteins, and their interrelationship with hormones, and the complexes they form in the cells under study. This involves the degree of concentration of the complexes formed, and the mechanism of the steroid hormone action with the receptor proteins. Much of the past work involved complexes of protein and steroid which were in the activated state. However, to obtain a true understanding of what is taking place in the cell itself requires a study of such complexes in the unactivated state as they appear in the natural cell itself, and from this, a better understanding of the biochemical mechanism of the process of forming the complex itself in its natural environment.
In the past, such complexes of hormones and receptor proteins were studies by affinity chromatography using an affinity gel-adsorbent prepared by cynogen bromide activation. However, the immobilized ligand leaks from their affinity matrix. This is a serious limitation for the use of affinity chromatography in the purification of hormone receptor proteins. An important improvement was achieved in the stability of the affinity adsorbent by attaching a substantially linear epoxy chain to the matrix, the former being terminated with an amino-functional group. Using such a technique, the possibility of leakage of the ligand was eliminated.
The steroid-receptor complex was obtained at an 46 percent overall yield, and the purification was about 1000-fold.
The following shows the synthesis route of the spacer arm, where M-(OH) is designated as the polymer matrix: ##STR4##
The following is the formula of the affinity gel-matrix coupled to the preferred hormone. ##STR5## R.sub.1 : H or OH or.dbd.O R.sub.2 : H or F
R.sub.3 : H or F PA1 R.sub.4 : OH or H PA1 R.sub.5 : OH or H ##STR6## R.sub.6 : H or CH.sub.3 X: 1 through 8 PA1 M: Matrix ##STR7## Alternative A ring with .DELTA.1,2 double bond.
The process or procedure of this invention is based on the biospecific adsorption of proteins to a steroid ligand attached to a substantially linear epoxy chain anchored on polysaccharide matrix.
In the process of the present invention, the affinity gel-adsorbent is contacted with the cytosol containing the receptor proteins which are bound to the latter gel-adsorbent. The gel-bound receptor is eluted, after separation from the cytosol, with (.sup.3 H) triamcinolone acetonide, and further purified by agarose gel-filtration. The protein yield obtained from the eluate was about 55 percent at 116-fold purification. However, when this was subjected to gel-filtration, the yield dropped to 46 percent but the purification of the proteins rose above 1000-fold.